Dictyostelium calmodulin: Production of a specific antiserum and localization in amoebae

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Abstract
A rabbit antiserum was raised against calmodulin from the eukaryotic microorganism Dictyostelium discoideum. In double immunodiffusion experiments, the antiserum formed an immunoprecipitation line with Dictyostelium calmodulin but not bovine brain calmodulin; competition radioimmunoassays showed no cross reactivity between the antiserum and calmodulins from bovine brain and spinach. The calmodulin content of vegetative Dictyostelium amoebae, determined by competition radioimmunoassay, was 0.5 μg/mg protein; similar levels were found in developing cells. The antiserum was used to visualize the distribution of calmodulin in Dictyostelium amoebae by indirect immunofluorescence. Cells were examined under various conditions: in suspension, attached to a substrate, and while phagocytosing yeast cells. In all cases, anticalmodulin staining was concentrated in the cell cortex. Parallel experiments using a monoclonal antibody against Dictyostelium myosin showed that this protein is also enriched in the cortical region of the cell.